Glucosylceramides (GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium (Pseudallescheria) boydii, one strain of Pseudallescheria ellipsoidea and one strain of P. angusta and purified by several chromatographic steps. Using high-performance thin layer chromatography (HPTLC), we found a similarity between GlcCer obtained from all of the analyzed strains. A detailed structural analysis of the P. ellipsoidea GlcCer was performed via electrospray ionization mass spectrometry (ESI-MS) and confirmed in 1- and 2-D heteronuclear NMR experiments (1H-13 C HSQC). GlcCer species produced by mycelial forms of these strains displayed the same structure previously demonstrated by our group for P. boydii, Cryptococcus neoformans, P. minustipora, Fusarium solani and Colletotrichum gloesporioides. A monoclonal antibody (mAb) against GlcCer was used for immunofluorescence experiments. Our results revealed that GlcCer is present on the surface of these fungi, and no difference was observed in the GlcCer structure of the present set of strains in terms of geographic or clinical origin, suggesting a conserved GlcCer structure similar to those previously described for S. apiospermum, S. aurantiacum and P. minutispora. The surface distribution of GlcCer in these fungi is suggestive of the involvement of this molecule in fungal growth.